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Mutant HSPB8 causes protein aggregates and a reduced mitochondrial membrane potential in dermal fibroblasts from distal hereditary motor neuropathy patients
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Neuromuscular Disorders 22 (2012) 699–711 www.elsevier.com/locate/nmd
Mutant HSPB8 causes protein aggregates and a reduced mitochondrial membrane potential in dermal fibroblasts from distal hereditary motor neuropathy patients Joy Irobi a,b, Anne Holmgren a,b, Vicky De Winter a,b, Bob Asselbergh a,b, Jan Gettemans c, Dirk Adriaensen d, Chantal Ceuterick-de Groote e, Rudy Van Coster f, Peter De Jonghe b,g,h, Vincent Timmerman a,b,⇑ a
Peripheral Neuropathy Group, Department of Molecular Genetics, VIB and University of Antwerp, Antwerpen 2610, Belgium b Laboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, Antwerpen 2610, Belgium c Department of Medical Protein Research, VIB and University of Ghent, Gent 9000, Belgium d Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerpen 2020, Belgium e Department of Ultrastructural Neuropathology, Institute Born-Bunge, University of Antwerp, Antwerpen 2610, Belgium f Department of Pediatrics, Division of Pediatric Neurology and Metabolism, Ghent University Hospital, Gent 9000, Belgium g Neurogenetics Group, Department of Molecular Genetics, VIB and University of Antwerp, Antwerpen 2610, Belgium h Division of Neurology, University Hospital of Antwerp, 2650 Edegem, Belgium Received 14 January 2012; received in revised form 23 March 2012; accepted 16 April 2012
Abstract Missense mutations in the small heat shock protein HSPB8 cause distal hereditary motor neuropathy (dHMN) and axonal Charcot-Marie-Tooth disease (CMT2L). We previously demonstrated that, despite the ubiquitous expression of HSPB8, motor neurons appear to be predominantly affected by HSPB8 mutations. Here, we studied the effect of mutant HSPB8 in primary fibroblast cultures derived from dHMN patients’ skin biopsy. In early passage cultures, we observed in all patients’ fibroblasts HSPB8 protein aggregates that were not detected in control cells. After applying heat shock stress on the patients’ early passage cultured cells, the protein aggregates coalesced into larger formations, while in control cells a homogenous upregulation of HSPB8 protein expression was seen. We also found a reduction in the mitochondrial membrane potential in the early passage cultures. After three months in culture, the number of cells with aggregates had become indistinguishable from that in controls and the mitochondrial membrane potential had returned to normal. These results emphasize the possible drawbacks of using patients’ non-neuronal cells to study neuropathological disease mechanisms. Ó 2012 Elsevier B.V. All rights reserved. Keywords: Hereditary motor neuropathy; Dermal biopsy; Small heat shock protein HSPB8; Protein aggregation and mitochondrial depolarization
1. Introduction Mutations in HSPB1 (HSP27) and HSPB8 (HSP22), two members of the small heat shock proteins (sHSP’s), ⇑ Corresponding author. Address: Peripheral Neuropathy Group, VIB Department of Molecular Genetics, University of Antwerp, Universiteitsplein 1, 2610 Antwerpen, Belgium. Tel.: +32 3 265 10 24; fax: +32 3 265 11 12. E-mail address: [email protected] (V. Timmerman).
0960-8966/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2012.04.005
are associated with inherited peripheral neuropathies [1,2]. So far, 16 mutations in HSPB1 were shown to cause either distal hereditary motor neuropathy (dHMN type 2b; OMIM 608634) or Charcot-Marie-Tooth type 2 (CMT2F; OMIM #606595) [3–9]. Two hot-spot mutations in HSPB8 cause dHMN type 2a (OMIM 158590) or CMT2L (OMIM 608673) [1,10]. So far, one report on a mutation in HSPB3 was shown in a single dHMN family [11] (OMIM 613376). Patients with mutant sHSP have a length-dependent degeneration of predominantly motor axons, which is defined as
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the dHMN phenotype [12]. However, if sensory nerves are affected the patients are classified as having axonal CMT2L or CMT2F [13]. The disease usually starts with paresis of the extensor muscles of the feet that rapidly progresses to weakness and occasionally paralysis of all distal muscles in the lower extremities within 5 years [12,14]. The HSPB1, HSPB8 and HSPB3 genes code for highly conserved stress proteins involved in multiple cellular processes [15–17]. These stress-induced molecular chaperones are vital for cell viability and protect the cells against environmental stress during aging by assisting in correct folding of denatured proteins and thus preventing aggregation of misfolded proteins [18]. The HSPB8 wild type protein was reported to inhibit the formation of aggregates in various cellular models for Huntington, Desmin-related cardiomyopathy and Alzheimer’s disease [19–22]. We have previously shown that HSPB8 mutations (K141N and K141E) target the hydrophobic strand of the conserved a-crystallin domain [1] which is essential for the structural and functional integrity of other sHSP’s [23,24]. In HSPB8, the mutated hot-spot lysine 141 amino acid corresponds to the arginine residues in other sHSP’s. When these positively charged residues are mutated in other sHSP’s, they result in various human disorders such as desmin-related myopathy (HSPB5-R120G, OMIM 608810), congenital cataract (HSPB4-R116C, OMIM 123580) and peripheral neuropathies (HSPB1-R140G, OMIM 606595 and HSPB8K141N/E, OMIM 158590) [1,5,23,25,26]. In a previous study we demonstrated that transduction with mutant HSPB8 (K141N and K141E) of primary mouse motor neurons resulted in spontaneous distal neurite degeneration without detectable signs of increased apoptosis [27]. Our in vitro studies showed that the HSPB8-K141N mutation enhances binding to the interacting partner HSPB1 and forms intracellular aggregates [1]. We hypothesized that mutant HSPB8 might enhance the formation of misfolded proteins and potentially interferes with normal cellular function in dHMN. To explore this hypothesis we obtained skin biopsies from patients with the HSPB8-K141N mutation. These patients belong to a multigeneration dHMN family in which we originally mapped the locus on chromosome 12q24 [28] and identified HSPB8 as the disease associated gene [1]. In this study, we cultured patients’ skin fibroblasts and examined the endogenous localization of the HSPB8 protein compared to control cultured fibroblasts. Interestingly, the early passage fibroblasts showed HSPB8 positive aggregates which were absent in later passage cell cultures. As accumulation of misfolded proteins may affect cell survival and disrupt mitochondrial function [29–31], we measured the mitochondrial inner membrane potential, the mitochondrial morphology and cell death. Although early passage cultured fibroblasts of dHMN patients showed a decrease in mitochondrial membrane potential, the mitochondrial morphology was not affected. Although ultrastructural examination of skin biopsy from dHMN showed axonal loss, patients’ fibroblasts did not show a drastic increase
in apoptosis, suggesting that dividing non-neuronal cells are capable of coping with the anomalous effects of mutant HSPB8. 2. Patients and methods 2.1. Subjects Skin biopsies were obtained from three normal controls (two women and one man) and two patients (two men) with dHMN type II carrying the K141N mutation in the small heat shock protein HSPB8. Clinical, electrophysiological and neuropathological characteristics of the patients with the HSPB8-K141N mutations were reported by Timmerman et al. [28] and re-examined by Dierick et al. [14]. Ages ranged from 36 to 50 years in control subjects used for fibroblast cultures and from 57 to 60 years in patients used for both cell cultures and electron microscopy examinations. Electron microscopy analysis of control skin biopsy is from a 53 year old control patient, selected after having reviewed nerve twigs from 14 control patients (51–74 years of age). Control samples were from patients with no evidence of peripheral neuropathy. These control patients were only suspected of having a neurometabolic disorder but were found to be normal by electron microscopy examination. Appropriate consent was obtained from participants and the Institutional Review Board from the University of Antwerp approved the study. 2.2. Skin biopsies and establishment of primary fibroblast cell cultures Five-millimeter punch forearm glabrous skin biopsies were obtained after local anesthesia, and were divided in two parts. One part was gluteraldehyde-fixed for electron microscopy analysis, while the other part were brought in 15 ml of F-12 (Ham) nutrient mixture + L-glutamine and kept overnight at 24 °C before further processing for cell culture. With a sterile disposable scalpel, the biopsies were chopped into 1 mm pieces, divided into two parts and cultured in a standard incubator at 37 °C, with 5% CO2 for 4– 5 weeks using complete culture medium: F-12 (Ham) nutrient mixture (Gibco, Invitrogen, supplemented with 15% fetal bovine serum, 1% penicilline/streptomicine, 1% glutamine). Primary fibroblast cells migrating out of the biopsies were monitored daily and every two days the culture medium was refreshed. Confluent culture dishes were passaged, fibroblast cells were further expanded and frozen in complete F12 medium supplemented with 10% DMSO. The rest of the primary cells were expanded and used for other experiments. 2.3. Immunocytochemistry of cultured cells For immunofluorescence, cells were grown to 75% confluence and then fixed in 3% paraformaldehyde at room temperature for 25 min, permeabilized with 0.1% Triton
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X-100 in phosphate-buffered saline (PBS) for 5 min, blocked in 1% BSA in PBS for 10 min and incubated with antibodies. To detect HSPB8, we used either polyclonal (1/ 250, Abcam) or monoclonal 2H5 (1/100, Abcam) antibodies to HSPB8. Double immunostainings were performed with polyclonal HSPB8 antibody co-stained with either HSP70 (1/1000, Abcam), or ubiquitin (1/200, Dako). Alexa488- and Alexa594-conjugated secondary antibodies (Invitrogen) were used and immunofluorescence images were obtained on a confocal microscope (LSM510 Meta; Zeiss) with a 40 or 63 Plan Apo objective using LSM 510 software. For heat shock treatment (24 h after seeding), cells were incubated in a water bath at 43 °C for 30 min and were allowed to recover for 24 h at 37 °C to overcome reduced expression due to heat-induced degradation of protein before proceeding with the immunostaining. 2.4. Measurement of the mitochondrial membrane potential and mitochondrial morphology The mitochondrial membrane potential (DWm) was measured with the fluorescent lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM, Invitrogen). Patient and control fibroblast cells were stained with TMRM (200 nM) for 30 min using a 37 °C warm water bath incubator. The TMRM fluorescence was analysed on a Partec CyFlow ML cytometer (Partec, Mu¨nster, Germany) and data were analysed with the Windows Multiple Document Interface software. Cell viability was checked using propidium iodide (PI; Sigma–Aldrich); and only PInegative cells were selected for DWm analysis. Different concentrations of the ionophore valinomycin (1 lM, 200 lM, 3 mM, 12 h incubation) was tested and used to induce the collapse of the membrane potentials which served as a control for loss of DWm. In each analysis, 10,000 events were recorded and each experiment was repeated three times. For mitochondrial morphologic analysis, fibroblast cells were cultivated in 35 mm glass-bottom dishes and when cells have reached the desired confluency, the medium was removed from the dishes and 100 nM MitoTrackerR Red CMXRos (Invitrogen) prewarmed (37 °C) probe was added, and cells were incubated for 30 min. Cells were imaged on a microlens-enhanced dual spinning disk confocal live cell imaging system (UltraVIEW; PerkinElmer). During imaging, cells were maintained at 37 °C and 5% CO2. Mitochondrial morphology was quantified by using a custom-made image analysis macro in the ImageJ software (http://rsbweb.nih.gov/ij/). Briefly, segmentation of the mitochondria (creation of a binary image mask) was performed by applying the default automatic threshold of ImageJ, after application of a median filter to reduce noise (1 pixel radius), reducing background with the rolling ball algorithm (20 pixel radius) and applying a 33 top-hat filter. The shape parameters (circularity and solidity) of individual mitochondria were collected by using the imageJ Particle Analyzer on all
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objects larger than 20 pixels. Both these output parameters vary between 0 and 1, with a value of 1 describing a perfect circle. This procedure is similar to the segmentation procedure and mitochondrial morphology analysis described by [32]. The mean circularity and solidity per image field (containing 1–5 cells) was calculated and at least six image fields were used to calculate the average circularity and solidity for each fibroblast line. 2.5. Detection of caspase-3 and -7 activity in cell-based assays The Caspase-GloÒ 3/7 Assay (Promega) is a luminescent assay that measures caspase-3 and -7 activity in cell cultures. An equal volume of reagent was added to approximately 20,000 cells of patient and control fibroblasts. Luminescence was measured after 30–90 min during incubation at room temperature using a microplate luminometer (model TD-20E, Turner design) with a 96 multiwell-plate format. Luminescence data were generated by combining the in triplicate results of two caspase-3 activity experiments. Culture medium without cells was used as a blank. As a positive control for the cell death assay, we used control fibroblasts treated for 3 h with 6 lM staurosporin. 2.6. Detection of Apo-BrdU-Red incorporation in DNA of cultured cells We used the Apo-BrdU-Red in situ DNA Fragmentation Assay Kit (BioVision, Gentaur) to measure internucleosomal DNA fragmentation as a hallmark of apoptosis in mammalian cells. Fibroblast cells were fixed with 3% formaldehyde in PBS and the DNA fragmentation assay was performed according to the manufacturer’s protocol. As a positive control for the cell death assay, we used control fibroblasts treated for 3 h with 3 lM staurosporin. 2.7. Western blotting Cells were lysed in NP40 lysis buffer (250 mM NaCl, 1 mM EDTA, 20 mM HEPES pH 7.9, 1% NP40, 20 mM b-glycerophosphate, 10 mM sodium fluoride, 4 mM sodium orthovanadate, 400 lg/ml sodium pyrophosphate, and 2 mM DDT) supplemented with protease inhibitor cocktail (Roche) for 20 min on ice and cleared by centrifugation. Cell extracts were boiled for 5 min and equal concentrations of cell lysates were resolved on a SDS– NuPage gel. The gel was transferred to a nitrocellulose membrane and processed for Western blotting as described previously [27,33]. For analysis of apoptosis, we used: cleaved-caspase 3, total-caspase 3, cleaved-caspase 9, total-caspase 9, cleaved-PARP and total-PARP (all antibodies were obtained from Cell Signalling and used at 1/1000 dilution). For analysis of protein expression, we used commercial available monoclonal HSPB8 antibody (1/100, Abcam) and anti-b-Actin (Sigma, 1/5000).
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2.8. Light and transmission electron microscopy Araldite blocks of glabrous dermal punch of two patients and controls were used for electron microscopy. Immersion fixation was achieved with 4% neutral buffered glutaraldehyde followed by 2% buffered osmium tetroxide and embedding in araldite [34]. Semithin sections were obtained with a Reichert Jung ultramicrotome (Leica, Wein, Austria) and collected on glass slides for light microscopy. Dermal myelinated nerve bundles were identified under the light microscope and were then trimmed and cut into ultrathin sections collected on copper grids. Sections were contrasted with uranyl acetate and lead citrate, and 34 cutaneous nerve bundles (6 nerves from patients; 28 nerves from control) were analyzed by a FEI CM10 transmission electron microscope (Philips, Eindhoven, The Netherlands) equipped with a goniometric coordinator. 3. Results 3.1. Distal HMN fibroblasts show transient HSPB8-positive protein aggregates In 2004, we reported that the HSPB8-K141N mutation results in perinuclear and cytoplasmic aggregates in transfected COS1 cells, and that these aggregates sequestered the HSPB1 protein [1]. As these experiments were based on transient transfection of mutant HSPB8 constructs, we could not exclude that the aggregates were due to possible overexpression of the mutant transcript. To exclude potential overexpression artifacts and to study the effect of mutant HSPB8 in dHMN patient cells we obtained skin biopsies from two related dHMN patients with the HSPB8-K141N mutation. Our findings were compared with skin biopsies from three unrelated healthy controls. From the skin biopsies we generated cultured fibroblasts, according to methods described. Gross morphological differences were not observed between cultured fibroblasts from the patients and control individuals. However, when we studied the cells by immunofluorescence microscopy using a HSPB8 directed antibody staining, every examined early passage primary fibroblast cell from dHMN patients showed multi-foci protein precipitates distributed throughout the cytoplasm. These HSPB8-positive aggregates were mainly detected in the early passage cultures (1–4 weeks) (Fig. 1A, patient). Primary fibroblasts from control individuals cultured under the same conditions mostly showed a homogeneous distribution of HSPB8-staining throughout the cytoplasm and nucleus (Fig. 1A, control). In later passage cultures (5–12 weeks), the mutant HSPB8 protein precipitates in patients’ cells coalesced into fewer cytoplasmic and/or perinuclear HSPB8positive aggregates and in some cells the protein accumulations were seen clustered around unknown structures in the cell (Fig. 1B). While protein aggregates in the early passage fibroblasts of dHMN patient were smaller in size and observed in every examined cell, those in the late passages were larger in diameter and scarcely observed (Fig. 1C).
After two months in culture, the percentage of aggregatepositive cells was still significantly higher in patients’ cells (data of three parallel experiments, n = 3 200 cells were counted: control HSPB8 wild type (WT) = 9.8% ± 1.52; patient 1-K141N = 37.6% ± 3, p-value t-test = 2.8 10 4; patient 2-K141N = 32.5% ± 14.7, p-value = 8.9 10 3). Next, we further examined this striking reduction in aggregate positive cells with increasing time in culture (Fig. 1C). To check whether the HSPB8 protein aggregates were targeted for degradation, we performed co-immunostainings with HSP70 and ubiquitin antibodies in dHMN fibroblasts and found that HSPB8-positive aggregates show enhanced HSP70 and ubiquitin protein localization (Fig. 2). These results indicate that fibroblasts containing HSPB8-protein aggregates are capable of recruiting the cellular machinery for elimination of either refolded or degraded misfolded HSPB8 proteins. However, we cannot exclude that selection of aggregate-negative cells in culture had contributed to the decrease in the percentage of aggregate-positive cells over time. Subsequently, we tested the effect of applying a heat shock (HS) stress on these cultured cells. We found that in the patients’ early passage cells multi-foci mutant HSPB8 precipitates coalesced into larger aggregates upon HS-stimulation, while control cells showed a homogenous upregulation of HSPB8 protein expression (Fig. 1D +HS). Protein expression of HSPB8 shown by Western blot was increased after heat shock, as was previously described [35,20], but no drastic changes were observed between patient and control fibroblasts (Fig. 1E). This study shows that upon heat shock stress the HSPB8 precipitates coalesced into larger protein aggregates which were subsequently cleared out as the passage time in cultures increased. Presumably mutant HSPB8 protein aggregates are cleared out with time in culture suggesting no cumulative effects and no major consequence in dividing fibroblast cells. 3.2. Mitochondrial membrane potential is reduced in early passage mutant fibroblasts Modulation of mitochondrial function and morphology is critical in neurodegenerative disorders [36]. Accumulation of misfolded proteins has been shown to disrupt mitochondrial function [37,38], plausibly by inducing defects in mitochondrial membrane potential (DWm) [39,40]. We first investigated whether there were changes in mitochondrial morphology by using live-cell imaging with cell-permeant MitoTracker Red CMXRos dye. The shape of the mitochondria was analyzed by measuring their average circularity and solidity. No differences could be detected in the mitochondrial shape descriptors between the control and patient fibroblasts (Supplementary figure 1). Absence of significant alterations in mitochondrial morphology does not exclude potential impairment of mitochondrial function. We further tested whether the fibroblast cells from patients exhibited a defect in mitochondrial energy metabolism. We measured DWm using a
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Fig. 1. HSPB8-K141N protein aggregates in dHMN skin fibroblasts. Primary fibroblast cells from skin biopsy of two patients and control persons were processed for immunofluorescence analysis. (A–B) The patients’ cells exhibit misfolded HSPB8 positive aggregates (patient, A and B, early and late passage). Such protein accumulations were absent in cells from control individuals (control, A and B). (C) The number of aggregate positive cells with increasing passage time in culture. (D) Heat shock stimulation of patients’ early passage cells (patient, D, +HS). Heat shock stimulation of control cells (control, D, +HS). (E) Western blot analysis of HSPB8 protein expression in patients and controls cell extracts. Selected images are representative for all patients and controls samples analyzed. No heat shock ( HS), with heat shock (+HS), nucleus is shown in blue, scale bar = 20 lm. Normalization was performed with b-actin.
fluorescent lipophilic cationic dye (TMRM) that accumulates within healthy polarized mitochondria. TMRM red fluorescence was measured by flow cytometry and analysis was performed in viable propidium iodide negative cells only. The depolarizing drug valinomycin was used as a positive control for loss of DWm (Fig. 3A, valinomycin). In early passage control cells, a high DWm (geometric mean value of 2 control samples = 397.1) was found demonstrating that the majority of the mitochondria were well polarized over the inner mitochondrial membrane (Fig. 3B and C, control 4 and 5). In contrast, when early passage patients’ cells were stained with TMRM, DWm was significantly lower (geometric mean value 2 patient samples = 272.6), suggesting that a significant portion of patient’s mitochondria were not adequately polarized (Fig. 3B and C, patient 1 and 2). (Mean data of three experiments, n = 3 10,000 cells per genotype were analysed: HSPB8-WT fibroblast control 4 = 433.49 ± 34; control 5 = 360.7 ± 18; patient 1-K141N = 251.6 ± 13,
p-value t-test = 5.8 10 3; patient 2-K141N = 293.5 ± 7, p-value = 1.5 10 2). When the same experiment was performed in late passage cells (after three months in culture) significant differences in DWm between patient and control groups were not observed (data not shown). These results suggest that the anomalous effects of mutant HSPB8 are well tolerated in dividing fibroblast cells. 3.3. Distal HMN fibroblasts do not show a major increase in apoptosis compared to control cells In many neurodegenerative diseases accumulation of misfolded proteins has been linked to cellular dysfunction and cell death [38,41]. Here, we investigated the relative survival of patient fibroblasts carrying the HSPB8K141N mutation by measuring the caspase activities. The apoptosis assays were only performed in late passage cells after two months in culture and not in early passage cells, since the early passage cultures were no longer available.
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Fig. 2. Co-localization of mutant HSPB8 protein aggregates with HSP70 and ubiquitin. Patient and control fibroblast cells were immunostained with antibodies against either HSP70 (green) or Ubiquitin (green) and HSPB8 (red). (A) Control fibroblast cells and (B) patient fibroblast cells both immunostained with HSP70 and HSPB8 antibodies. (C) Control fibroblast cells and (D) patient fibroblast cells both immunostained with antibodies to ubiquitin and HSPB8. Selected images are representative for all patients and controls samples analysed. Nucleus is shown in blue, Scale bar = 10 lm.
We observed an increase of caspase 3/7 activity in fibroblasts of patient 2 but not of patient 1 as compared to control fibroblasts (Fig. 4A, patient 2 vs. control 3 pvalue = 0.037, patient 2 vs. control 5 p-value = 0.057. Patient 1 vs. control 3 p-value = 0.138, patient 1 vs. control 5 p-value = 0.247). In addition, we investigated the distribution of internucleosomal DNA fragmentation as a hallmark of apoptosis [42]. Staurosporin treatment was used
as a positive control to induce apoptosis (Fig. 4B). Control fibroblasts were Br-dUTP negative (Fig. 4C). The patients’ fibroblast cultures contained more Br-dUTP positive cells compared to control cultures (Fig. 4D and E). Furthermore, protein levels of three well established apoptotic markers were evaluated in late passage cells after three months in culture: cleaved-caspase 3, cleaved-caspase 9 and cleaved-poly (ADP-ribose) polymerase (PARP)
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Fig. 3. Loss of mitochondrial membrane potential in patient’s fibroblasts. Mitochondrial membrane potential (DWm) was measured in cultured patients and controls fibroblasts. (A) Valinomycin treated cells were used as a positive control for loss of DWm. (B and C) TMRM histogram analysis of DWm of 2 patients and 2 control fibroblast cells. (D) Statistical analysis (DWm geometric mean and stdev) was performed on three different experiments.
[43,44]. There was no clear evidence of accumulation of these apoptotic markers in either patients or controls cell lysates in two different experiments (Fig. 5). Taken together, these results indicate that the HSPB8-K141N mutation could elevate the vulnerability to pro-apoptotic stimuli in primary fibroblast cultures, although not all of used apoptosis markers confirm these results. 3.4. Cutaneous nerves show axonal loss in dHMN patients Since we were unable to obtain post-mortem spinal cord or motor nerve biopsy samples from dHMN patients with the HSPB8-K141N mutation to test the presence of intracellular protein aggregates, we studied skin biopsy cutaneous nerves from two dHMN patients [28] by electron microscopy. This approach constitutes a minimal invasive procedure and is also used to monitor the progression of the neuropathy and regeneration of nerve fibers [45,46]. Electron microscopy of cutaneous nerve fascicles revealed a decreased number of myelinated and unmyelinated axons in both patients (Fig. 6A and C), in comparison with agematched control individuals such as illustrated in a dermal nerve bundle of a 53-year old control (Fig. 6B). Few formations with slight axonal swellings, suggestive of axonal regeneration sprouting (not shown), and a thinly myelinated (regenerated) axon were detected (Fig 6C). Denervated Schwann cells forming collagen pockets (Fig 6D) and large bundles of endoneurial collagen fibrils (Fig 6A), were observed. Ultrastructural analysis did not reveal obvious protein inclusions, neither did it reveal alterations of the mitochondrial morphology, nor onion-bulb-like
formations in the patients’ cutaneous nerve fascicles. These results suggest that the mutation in HSPB8 affects sensory axons but only to a minor extent. 4. Discussion HSPB8 belongs to the family of small heat shock proteins. These proteins are multifunctional molecular chaperones that participate in multiple cellular processes [47–49]. Although HSPB8 is expressed in many tissues [20,50], the expression level is higher in the peripheral nervous system [1,35] which explains why mutations in HSPB8 cause dHMN and CMT2 neuropathy [1,10]. Previously, we demonstrated that rodent motor neuron cultures transiently expressing mutant HSPB8 protein resulted in distal neurite degeneration without apoptosis [27]. In a former work, we showed that overexpression of mutant HSPB8 resulted in intracellular protein aggregates [1]. Here, we show that early passage fibroblast cells from two related dHMN patients have an accumulation of HSPB8 protein aggregates and a decrease in mitochondrial membrane potential. These protein aggregates were cleared out after three months in culture and patient cells became indistinguishable from controls. We also observed that under stress conditions endogenous mutant HSPB8 protein induced intracellular protein aggregates in cultured skin fibroblasts from the dHMN patients. Such accumulation of protein was not seen in three fibroblasts from unrelated healthy individuals. Heat shock stress stimulation of patients’ early passage cells resulted in larger accumulations of HSPB8 positive
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Fig. 4. Patient fibroblast cells do not elicit marked apoptosis. Cell death assay of: (A) dHMN patients and controls fibroblast cells. As positive control, cells were treated with staurosporin, and cell death luminescence was measured. Data are presented as means ± stdev of three different experiments. (B–D) Apoptosis assay of patients and control fibroblast cells were processed for DNA fragmentation followed by immunofluorescence. (B) Control fibroblasts were treated with staurosporin as positive control for cell death and immunostained with HSPB8 antibody (green) and processed for DNA fragmentation (red). (C) Control fibroblasts. (D) Patient fibroblasts. (E) The quantification of DNA fragmentation in patient and control fibroblasts. Selected images are representative for all patient and control samples analyzed. Scale bar = 30 lm. Statistical significance denoted with a star (*p-value <0.05) was determined using Student’s t-test.
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Fig. 5. Mutant fibroblasts do not result in increased activation of apoptosis. Activation of enzymes implicated in initiation and execution of apoptosis was examined by Western blot of cleaved (17 and 19 kDa) and total caspase 3 (35 kDa), cleaved (35 and 37 kDa) and total caspase 9 (47 kDa) and cleaved (89 kDa) and total PARP (116 kDa) in patients and controls fibroblast cells. Selected images are representative for two different western blot experiments. Normalization was performed with b-actin.
precipitates into aggresome-like structures and not in a homogenous upregulation of HSPB8 protein as observed in control cells. An accumulation of more protein aggregates suggests that the mutant protein is associated with a defect in stress-induced molecular chaperones that are considered as vital for cell viability. Evidently, in the endoplasmic reticulum proteins that fail to fold are destroyed.
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Such misfolded proteins are transported to the cytosol where ubiquitination and finally proteasomal proteolysis dispense with the unwanted polypeptides [51]. Our data showed that the HSPB8 protein aggregates tested positive for both HSP70 and ubiquitin, suggesting that the protein accumulations are being degraded by proteasomal proteolysis. We cannot exclude the possibility of other mechanisms for intracellular protein degradation being activated. In addition to its canonical function as a molecular chaperone, HSPB8 acts in association with the cochaperone BAG3 to target protein aggregates for destruction via autophagy [52–54]. However, in a recent report it was shown that overexpression of the HSPB8-K141N mutant protein failed to stimulate autophagy [55]. In any case, the observation that the number of mutant HSPB8 cells with protein aggregates decreases with an increase in passage time, points to a stress induction at the start of the culture that causes mutant HSPB8 to aggregate, and a subsequent clearance of these aggregates during the time in culture or alternatively, a selection of aggregate-free cells. We can speculate that stress caused by the biopsy procedure and adaptation to cell culture conditions caused a transient HSPB8 upregulation, which resulted in aggregation of the mutant protein. Refolding of misfolded protein could deplete cellular energy pools that are needed for normal cellular functions, especially in a confined axoplasmic region. Mitochondrial dysfunction plays a pivotal role in contributing to defective energy metabolism and neuronal degeneration [56]. We tested whether the fibroblast cells from patients exhibit any defect in mitochondrial energy metabolism using TMRM, a mitochondria transmembrane potential (DWm) marker. We did not observe obvious changes in mitochondrial morphology but detected a significant reduction in DWm in the patients’ fibroblast cells as compared to control cells. These data suggest that dHMN fibroblasts have more depolarized mitochondria than control fibroblasts. An extrapolation of the observations made in patients’ cultured fibroblasts to the nervous system should be interpreted with caution but might explain some of the findings in the nervous system, especially in the motor neurons with long axons. It is possible that a reduction of the mitochondrial DWm is well tolerated in the cells without axons, but could become detrimental in the cells with long axons. Accumulation of depolarized mitochondria in a late onset disease could disrupt cellular homeostasis and trigger synaptic and axoplasmic dysfunctions [38]. In the present study, cells of patient primary fibroblast show increased incidence of apoptosis based on some of the markers we used. In a former work however, we studied the significance of HSPB8 mediated cell death in primary rodent ventral horn cultures after four days of mutant protein transduction and found cell type specific neurite defects, but no signs of defective apoptosis in all studied cell types [27]. Given that axonal and not neuronal loss is the first manifestation of the disease in patients [28,57], we do not assume that upregulation of apoptotic signaling
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Fig. 6. Evidence of axonal loss in skin biopsy of patients with the HSPB8-K141N mutation. Electron micrographs of cutaneous nerve fascicles. General views revealing a decreased number of myelinated and unmyelinated axons in both patients (A and C) in comparison with a control individual (B). Note obvious endoneurial collagen formation (arrows) in (A) and a thinly myelinated (regenerating) axon (arrow) (C). Higher magnification of denervated Schwann cells with collagen pockets (arrows) of the nerve illustrated in C (D) while several normal unmyelinated axons () within Schwann cell (arrows) cytoplasms, are shown in (E). Scale bars = 1 lm.
is a primary mechanism for mutant HSPB8 pathogenicity. We consider that the increased susceptibility to apoptosis in the mutant HSPB8 cultures is a secondary and more general effect in the mutant HSPB8 fibroblasts that results from their defective capacity to handle cellular stress. Overall, our results demonstrate that although patients’ fibroblast cells exhibited subtle signs of stress, these cells were able to tolerate the anomalous effects of mutant HSPB8. We speculate that accumulation of such subtle signs of stress might be well tolerated in the neuronal cell body but plausibly could contribute to the process of axonal loss in dHMN. Corroborating our reasoning, it was suggested in a recent report that if a neuropathy takes years to develop, the mutants that cause such a neuropathy would be expected to have relatively subtle defects and only with time are the cumulative effects of the mutation on cellular function likely to become fully apparent [58]. Notably, the lack of dramatic effects on apoptosis in patients’
cells is in accordance with the dHMN disease phenotype, being debilitating but not life threatening. As dHMN is predominantly a motor neuropathy, it would be ideal to study motor nerves but we were unable to obtain motor nerve autopsy material. Motor nerves are also difficult to obtain by biopsy because it would cause a motor deficit in the corresponding muscles. The only nerves that were available for investigation were sensory cutaneous nerves in a skin biopsy specimen from the two patients carrying the HSPB8 K141N mutation. Ultrastructural analysis carried out by transmission electron microscopy [28] revealed that patients’ cutaneous nerves showed clear signs of axonal loss and slight axonal regeneration. We cannot exclude the possibility that these findings might be due to age-related changes [59] although such ultrastructural findings were not detected in the investigated control skin biopsies. We did not observe any obvious protein inclusion or abnormal mitochondrial shape in the skin
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biopsies from the patients. Overall, our results indicate that mutant HSPB8 induces only minor degeneration of sensory nerve axons in dHMN patients’ cutaneous nerves. This confirms that, although the brunt of the injury in motor neuropathies falls on the motor neurons, some minor sensory involvement can be detected. Of note is that the K141N mutation is not only associated to dHMN but also to CMT2 phenotype [10]. By using skin biopsies and derived fibroblasts to study dHMN, a disease that primarily affects motor neurons, we are obviously using a suboptimal disease model, especially since we have shown that the pathogenic effect of the HSPB8 mutation is specifically affecting motor neurons [27]. In this regard, ultrastructural examination of cutaneous nerve fascicles allows examining sensory nerves rather than motor nerves (Fig. 6). In addition, since biopsies of the relevant patients are relatively scarce, it was not feasible to include larger numbers of individuals in this study to further ascertain our findings. Moreover, during the course of this study we were confronted with the observation that in the initial passages of the fibroblast cultures, the majority of cells showed mutant HSPB8 aggregates, whereas after some time in culture the prevalence of HSPB8 aggregate positive fibroblasts decreased unexpectedly. Because it was not possible to resample the same patients, mainly for deontological reasons, we could not reassess the patient fibroblasts in their initial state to replicate our experiments and to further explore this phenomenon experimentally. However, there are also benefits in using this approach compared to alternative strategies. (i) It allows the examination of patient’s endogenous mutant proteins without the complications of overexpression artifacts (Fig. 1). (ii) It can reveal damage or morphological alterations in small dermal nerve fascicles (Fig. 6), and in contrast to conventional peripheral nerve biopsy a skin biopsy is less invasive and is easier to perform [46]. (iii) Skin biopsy fibroblasts can be reprogrammed to generate patient-specific induced pluripotent stem cells and neural progenitor cells (iPS and NPC), which can be used to reveal novel biological insights which can, in turn, lead to clinical benefits [60]. In conclusion our work demonstrates that early passage primary fibroblasts from dHMN patients show abnormal changes including the presence of protein aggregates, a decrease of the inner mitochondrial potential and a reduction in cell viability. These defects were not observed in late passage primary cells, which emphasizes the complexity and inadequacy of studying a neuropathy by using nonneuronal patient material. Direct reprogramming of the patients’ fibroblasts to iPS cell-derived neurons could provide a platform to understand the effect of mutant sHSP’s in human motor neurons and could reveal reliable biomarkers for disease prevention strategies. Acknowledgments We are grateful to the patients and control individuals who provided nerve and skin biopsies. The authors thank
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G. Munteanu, K.G. Claeys, C. Michiels, N. Cools, M. Lenjuo, V. De Corte, B. Van Loo, J. Smet and N. Festjens for technical assistance. We thank J. Wauters, J. Lambert, R. Bendorf, K. Verhoeven, V. Van Tendeloo for discussions and exchange of expertise. We thank L. Svensson, J. Van Daele, D. De Rijck, L. De Wit and I. Bats for technical assistance with electron and confocal microscopy. This work was supported by the Methusalem and Hercules (AUAH-09-001) programs of the Flemish Government, the Fund for Scientific Research (FWO-Flanders), the Medical Foundation Queen Elisabeth, the “Association Belge contre les Maladies Neuromusculaires”, the American Muscular Dystrophy Association, and the Interuniversity Attraction Poles P6/43 program of the Belgian Federal Science Policy Office. A.H. is supported by a Ph.D. fellowship of the Institute for Science and Technology (IWT). J.I. is postdoctoral researcher supported by the FWOFlanders. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.nmd.2012.04.005. References [1] Irobi J, Van Impe K, Seeman P, et al. Hot-spot residue in small heatshock protein 22 causes distal motor neuropathy. Nat Genet 2004;36:597–601. [2] Evgrafov OV, Mersiyanova I, Irobi J, et al. Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. Nat Genet 2004;36:602–6. [3] Rossor AM, Kalmar B, Greensmith L, Reilly MM. The distal hereditary motor neuropathies. J Neurol Neurosurg Psychiatry 2012;83:6–14. [4] Kijima K, Numakura C, Goto T, et al. Small heat shock protein 27 mutation in a Japanese patient with distal hereditary motor neuropathy. J Hum Genet 2005;50:473–6. [5] Houlden H, Laura M, Wavrant-DeVrieze F, et al. Mutations in the HSP27 (HSPB1) gene cause dominant, recessive, and sporadic distal HMN/CMT type 2. Neurology 2008;71:1660–8. [6] James PA, Rankin J, Talbot K. Asymmetrical late onset motor neuropathy associated with a novel mutation in the small heat shock protein HSPB1 (HSP27). J Neurol Neurosurg Psychiatry 2008;79:461–3. [7] Luigetti M, Fabrizi GM, Madia F, et al. A novel HSPB1 mutation in an Italian patient with CMT2/dHMN phenotype. J Neurol Sci 2010;298:114–7. [8] Capponi S, Geroldi A, Fossa P, et al. HSPB1 and HSPB8 in inherited neuropathies: study of an Italian cohort of dHMN and CMT2 patients. J Peripher Nerv Syst 2011;16:287–94. [9] Mandich P, Grandis M, Varese A, et al. Severe neuropathy after diphtheria-tetanus-pertussis vaccination in a child carrying a novel frame-shift mutation in the small heat-shock protein 27 gene. J Child Neurol 2010;25:107–9. [10] Tang BS, Zhao GH, Luo W, et al. Small heat-shock protein 22 mutated in autosomal dominant Charcot-Marie-Tooth disease type 2L. Hum Genet 2005;116:222–4. [11] Kolb SJ, Snyder PJ, Poi EJ, et al. Mutant small heat shock protein B3 causes motor neuropathy: utility of a candidate gene approach. Neurology 2010;74:502–6.
710
J. Irobi et al. / Neuromuscular Disorders 22 (2012) 699–711
[12] Harding AE, Thomas PK. Hereditary distal spinal muscular atrophy. A report on 34 cases and a review of the literature. J Neurol Sci 1980;45:337–48. [13] Saporta AS, Sottile SL, Miller LJ, et al. Charcot-Marie-Tooth disease subtypes and genetic testing strategies. Ann Neurol 2011; 69:22–33. [14] Dierick I, Baets J, Irobi J, et al. Relative contribution of mutations in genes for autosomal dominant distal hereditary motor neuropathies: a genotype-phenotype correlation study. Brain 2008;131:1217–27. [15] Haslbeck M, Franzmann T, Weinfurtner D, Buchner J. Some like it hot: the structure and function of small heat-shock proteins. Nat Struct Mol Biol 2005;12:842–6. [16] Drew AP, Blair IP, Nicholson GA. Molecular genetics and mechanisms of disease in distal hereditary motor neuropathies: insights directing future genetic studies. Curr Mol Med 2011. [17] Dierick I, Irobi J, De Jonghe P, Timmerman V. Small heat shock proteins in inherited peripheral neuropathies. Ann Med 2005;37:413–22. [18] Rothman JE, Schekman R. Molecular mechanism of protein folding in the cell. Cell 2011. [19] Carra S, Sivilotti M, Chavez Zobel AT, Lambert H, Landry J. HspB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells. Hum Mol Genet 2005;14:1659–69. [20] Chowdary TK, Raman B, Ramakrishna T, Rao CM. Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperonelike activity. Biochem J 2004;381:379–87. [21] Wilhelmus MM, Boelens WC, Otte-Holler I, et al. Small heat shock proteins inhibit amyloid-beta protein aggregation and cerebrovascular amyloid-beta protein toxicity. Brain Res 2006;1089:67–78. [22] Wilhelmus MM, Otte-Holler I, Wesseling P, et al. Specific association of small heat shock proteins with the pathological hallmarks of Alzheimer’s disease brains. Neuropathol Appl Neurobiol 2006;32:119–30. [23] Bera S, Thampi P, Cho WJ, Abraham EC. A positive charge preservation at position 116 of alpha A-crystallin is critical for its structural and functional integrity. Biochemistry 2002;41:12421–6. [24] Kasakov AS, Bukach OV, Seit-Nebi AS, Marston SB, Gusev NB. Effect of mutations in the beta5–beta7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11). FEBS J 2007;274:5628–42. [25] Litt M, Kramer P, LaMorticella DM, et al. Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystallin gene CRYAA. Hum Mol Genet 1998;7:471–4. [26] Vicart P, Caron A, Guicheney P, et al. A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat Genet 1998;20:92–5. [27] Irobi J, Almeida-Souza L, Asselbergh B, et al. Mutant HSPB8 causes motor neuron-specific neurite degeneration. Hum Mol Genet 2010;19:3254–65. [28] Timmerman V, Raeymaekers P, Nelis E, et al. Linkage analysis of distal hereditary motor neuropathy type II (distal HMN II) in a single pedigree. J Neurol Sci 1992;109:41–8. [29] Pedrini S, Sau D, Guareschi S, et al. ALS-linked mutant SOD1 damages mitochondria by promoting conformational changes in Bcl2. Hum Mol Genet 2010;19:2974–86. [30] Caspersen C, Wang N, Yao J, et al. Mitochondrial Abeta: a potential focal point for neuronal metabolic dysfunction in Alzheimer’s disease. FASEB J 2005;19:2040–1. [31] Hauptmann S, Keil U, Scherping I, et al. Mitochondrial dysfunction in sporadic and genetic Alzheimer’s disease. Exp Gerontol 2006;41:668–73. [32] Koopman WJ, Verkaart S, Visch HJ, et al. Inhibition of complex I of the electron transport chain causes O2 -mediated mitochondrial outgrowth. Am J Physiol Cell Physiol 2005;288:C1440–50. [33] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76:4350–4.
[34] Ceuterick C, Martin JJ. Electron microscopic features of skin in neurometabolic disorders. J Neurol Sci 1992;112:15–29. [35] Kirbach BB, Golenhofen N. Differential expression and induction of small heat shock proteins in rat brain and cultured hippocampal neurons. J Neurosci Res 2011;89:162–75. [36] Kieper N, Holmstrom KM, Ciceri D, et al. Modulation of mitochondrial function and morphology by interaction of Omi/ HtrA2 with the mitochondrial fusion factor OPA1. Exp Cell Res 2010;316:1213–24. [37] Winklhofer KF, Tatzelt J, Haass C. The two faces of protein misfolding: gain- and loss-of-function in neurodegenerative diseases. EMBO J 2008;27:336–49. [38] Schon EA, Manfredi G. Neuronal degeneration and mitochondrial dysfunction. J Clin Invest 2003;111:303–12. [39] Correia SC, Santos RX, Perry G, et al. Mitochondria: the missing link between preconditioning and neuroprotection. J Alzheimers Dis 2010;20(Suppl. 2):S475–85. [40] Balaban RS, Nemoto S, Finkel T. Mitochondria, oxidants, and aging. Cell 2005;120:483–95. [41] Hoffstrom BG, Kaplan A, Letso R, et al. Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins. Nat Chem Biol 2010;6:900–6. [42] Saraste A, Pulkki K. Morphologic and biochemical hallmarks of apoptosis. Cardiovasc Res 2000;45:528–37. [43] Kroemer G, Galluzzi L, Vandenabeele P, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 2009;16:3–11. [44] Vanden Berghe T, Denecker G, Brouckaert G, et al. More than one way to die: methods to determine TNF-induced apoptosis and necrosis. Methods Mol Med 2004;98:101–26. [45] Ceuterick-De GC, De Jonghe P, Timmerman V, et al. Infantile demyelinating neuropathy associated with a de novo point mutation on Ser72 in PMP22 and basal lamina onion bulbs in skin biopsy. Pathol Res Pract 2001;197:193–8. [46] Li J, Bai Y, Ghandour K, et al. Skin biopsies in myelin-related neuropathies: bringing molecular pathology to the bedside. Brain 2005;128:1168–77. [47] Arrigo AP, Paul C, Ducasse C, et al. Small stress proteins: novel negative modulators of apoptosis induced independently of reactive oxygen species. Prog Mol Subcell Biol 2002;28:185–204. [48] Charette SJ, Landry J. The interaction of HSP27 with Daxx identifies a potential regulatory role of HSP27 in Fas-induced apoptosis. Ann N Y Acad Sci 2000;926:126–31. [49] Huot J, Houle F, Spitz DR, Landry J. HSP27 phosphorylationmediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res 1996;56:273–9. [50] Benndorf R, Sun X, Gilmont RR, et al. HSP22, a new member of the small heat shock protein superfamily, interacts with mimic of phosphorylated HSP27 ((3D)HSP27). J Biol Chem 2001;276:26753–61. [51] Lilley BN, Ploegh HL. A membrane protein required for dislocation of misfolded proteins from the ER. Nature 2004;429:834–40. [52] Crippa V, Sau D, Rusmini P, et al. The small heat shock protein B8 (HspB8) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS). Hum Mol Genet 2010;19:3440–56. [53] Arndt V, Dick N, Tawo R, et al. Chaperone-assisted selective autophagy is essential for muscle maintenance. Curr Biol 2010;20:143–8. [54] Carra S, Seguin SJ, Landry J. HspB8 and Bag3: a new chaperone complex targeting misfolded proteins to macroautophagy. Autophagy 2008;4:237–9. [55] Kwok AS, Phadwal K, Turner BJ, et al. HspB8 mutation causing hereditary distal motor neuropathy impairs lysosomal delivery of autophagosomes. J Neurochem 2011. [56] Eckert A, Keil U, Marques CA, et al. Mitochondrial dysfunction, apoptotic cell death, and Alzheimer’s disease. Biochem Pharmacol 2003;66:1627–34.
J. Irobi et al. / Neuromuscular Disorders 22 (2012) 699–711 [57] Timmerman V, Beuten J, Irobi J, et al. Distal hereditary motor neuropathy type II (distal HMN type II): phenotype and molecular genetics. Ann N Y Acad Sci 1999;883:60–4. [58] Holzbaur EL, Scherer SS. Microtubules, axonal transport, and neuropathy. N Engl J Med 2011;365:2330–2.
711
[59] Timmerman V, De Jonghe P, Simokovic S, et al. Distal hereditary motor neuropathy type II (distal HMN II): mapping of a locus to chromosome 12q24. Hum Mol Genet 1996;5:1065–9. [60] Han SS, Williams LA, Eggan KC. Constructing and deconstructing stem cell models of neurological disease. Neuron 2011;70:626–44.
Neuromuscular Disorders 22 (2012) 699–711 www.elsevier.com/locate/nmd
Mutant HSPB8 causes protein aggregates and a reduced mitochondrial membrane potential in dermal fibroblasts from distal hereditary motor neuropathy patients Joy Irobi a,b, Anne Holmgren a,b, Vicky De Winter a,b, Bob Asselbergh a,b, Jan Gettemans c, Dirk Adriaensen d, Chantal Ceuterick-de Groote e, Rudy Van Coster f, Peter De Jonghe b,g,h, Vincent Timmerman a,b,⇑ a
Peripheral Neuropathy Group, Department of Molecular Genetics, VIB and University of Antwerp, Antwerpen 2610, Belgium b Laboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, Antwerpen 2610, Belgium c Department of Medical Protein Research, VIB and University of Ghent, Gent 9000, Belgium d Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, Antwerpen 2020, Belgium e Department of Ultrastructural Neuropathology, Institute Born-Bunge, University of Antwerp, Antwerpen 2610, Belgium f Department of Pediatrics, Division of Pediatric Neurology and Metabolism, Ghent University Hospital, Gent 9000, Belgium g Neurogenetics Group, Department of Molecular Genetics, VIB and University of Antwerp, Antwerpen 2610, Belgium h Division of Neurology, University Hospital of Antwerp, 2650 Edegem, Belgium Received 14 January 2012; received in revised form 23 March 2012; accepted 16 April 2012
Abstract Missense mutations in the small heat shock protein HSPB8 cause distal hereditary motor neuropathy (dHMN) and axonal Charcot-Marie-Tooth disease (CMT2L). We previously demonstrated that, despite the ubiquitous expression of HSPB8, motor neurons appear to be predominantly affected by HSPB8 mutations. Here, we studied the effect of mutant HSPB8 in primary fibroblast cultures derived from dHMN patients’ skin biopsy. In early passage cultures, we observed in all patients’ fibroblasts HSPB8 protein aggregates that were not detected in control cells. After applying heat shock stress on the patients’ early passage cultured cells, the protein aggregates coalesced into larger formations, while in control cells a homogenous upregulation of HSPB8 protein expression was seen. We also found a reduction in the mitochondrial membrane potential in the early passage cultures. After three months in culture, the number of cells with aggregates had become indistinguishable from that in controls and the mitochondrial membrane potential had returned to normal. These results emphasize the possible drawbacks of using patients’ non-neuronal cells to study neuropathological disease mechanisms. Ó 2012 Elsevier B.V. All rights reserved. Keywords: Hereditary motor neuropathy; Dermal biopsy; Small heat shock protein HSPB8; Protein aggregation and mitochondrial depolarization
1. Introduction Mutations in HSPB1 (HSP27) and HSPB8 (HSP22), two members of the small heat shock proteins (sHSP’s), ⇑ Corresponding author. Address: Peripheral Neuropathy Group, VIB Department of Molecular Genetics, University of Antwerp, Universiteitsplein 1, 2610 Antwerpen, Belgium. Tel.: +32 3 265 10 24; fax: +32 3 265 11 12. E-mail address: [email protected] (V. Timmerman).
0960-8966/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2012.04.005
are associated with inherited peripheral neuropathies [1,2]. So far, 16 mutations in HSPB1 were shown to cause either distal hereditary motor neuropathy (dHMN type 2b; OMIM 608634) or Charcot-Marie-Tooth type 2 (CMT2F; OMIM #606595) [3–9]. Two hot-spot mutations in HSPB8 cause dHMN type 2a (OMIM 158590) or CMT2L (OMIM 608673) [1,10]. So far, one report on a mutation in HSPB3 was shown in a single dHMN family [11] (OMIM 613376). Patients with mutant sHSP have a length-dependent degeneration of predominantly motor axons, which is defined as
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the dHMN phenotype [12]. However, if sensory nerves are affected the patients are classified as having axonal CMT2L or CMT2F [13]. The disease usually starts with paresis of the extensor muscles of the feet that rapidly progresses to weakness and occasionally paralysis of all distal muscles in the lower extremities within 5 years [12,14]. The HSPB1, HSPB8 and HSPB3 genes code for highly conserved stress proteins involved in multiple cellular processes [15–17]. These stress-induced molecular chaperones are vital for cell viability and protect the cells against environmental stress during aging by assisting in correct folding of denatured proteins and thus preventing aggregation of misfolded proteins [18]. The HSPB8 wild type protein was reported to inhibit the formation of aggregates in various cellular models for Huntington, Desmin-related cardiomyopathy and Alzheimer’s disease [19–22]. We have previously shown that HSPB8 mutations (K141N and K141E) target the hydrophobic strand of the conserved a-crystallin domain [1] which is essential for the structural and functional integrity of other sHSP’s [23,24]. In HSPB8, the mutated hot-spot lysine 141 amino acid corresponds to the arginine residues in other sHSP’s. When these positively charged residues are mutated in other sHSP’s, they result in various human disorders such as desmin-related myopathy (HSPB5-R120G, OMIM 608810), congenital cataract (HSPB4-R116C, OMIM 123580) and peripheral neuropathies (HSPB1-R140G, OMIM 606595 and HSPB8K141N/E, OMIM 158590) [1,5,23,25,26]. In a previous study we demonstrated that transduction with mutant HSPB8 (K141N and K141E) of primary mouse motor neurons resulted in spontaneous distal neurite degeneration without detectable signs of increased apoptosis [27]. Our in vitro studies showed that the HSPB8-K141N mutation enhances binding to the interacting partner HSPB1 and forms intracellular aggregates [1]. We hypothesized that mutant HSPB8 might enhance the formation of misfolded proteins and potentially interferes with normal cellular function in dHMN. To explore this hypothesis we obtained skin biopsies from patients with the HSPB8-K141N mutation. These patients belong to a multigeneration dHMN family in which we originally mapped the locus on chromosome 12q24 [28] and identified HSPB8 as the disease associated gene [1]. In this study, we cultured patients’ skin fibroblasts and examined the endogenous localization of the HSPB8 protein compared to control cultured fibroblasts. Interestingly, the early passage fibroblasts showed HSPB8 positive aggregates which were absent in later passage cell cultures. As accumulation of misfolded proteins may affect cell survival and disrupt mitochondrial function [29–31], we measured the mitochondrial inner membrane potential, the mitochondrial morphology and cell death. Although early passage cultured fibroblasts of dHMN patients showed a decrease in mitochondrial membrane potential, the mitochondrial morphology was not affected. Although ultrastructural examination of skin biopsy from dHMN showed axonal loss, patients’ fibroblasts did not show a drastic increase
in apoptosis, suggesting that dividing non-neuronal cells are capable of coping with the anomalous effects of mutant HSPB8. 2. Patients and methods 2.1. Subjects Skin biopsies were obtained from three normal controls (two women and one man) and two patients (two men) with dHMN type II carrying the K141N mutation in the small heat shock protein HSPB8. Clinical, electrophysiological and neuropathological characteristics of the patients with the HSPB8-K141N mutations were reported by Timmerman et al. [28] and re-examined by Dierick et al. [14]. Ages ranged from 36 to 50 years in control subjects used for fibroblast cultures and from 57 to 60 years in patients used for both cell cultures and electron microscopy examinations. Electron microscopy analysis of control skin biopsy is from a 53 year old control patient, selected after having reviewed nerve twigs from 14 control patients (51–74 years of age). Control samples were from patients with no evidence of peripheral neuropathy. These control patients were only suspected of having a neurometabolic disorder but were found to be normal by electron microscopy examination. Appropriate consent was obtained from participants and the Institutional Review Board from the University of Antwerp approved the study. 2.2. Skin biopsies and establishment of primary fibroblast cell cultures Five-millimeter punch forearm glabrous skin biopsies were obtained after local anesthesia, and were divided in two parts. One part was gluteraldehyde-fixed for electron microscopy analysis, while the other part were brought in 15 ml of F-12 (Ham) nutrient mixture + L-glutamine and kept overnight at 24 °C before further processing for cell culture. With a sterile disposable scalpel, the biopsies were chopped into 1 mm pieces, divided into two parts and cultured in a standard incubator at 37 °C, with 5% CO2 for 4– 5 weeks using complete culture medium: F-12 (Ham) nutrient mixture (Gibco, Invitrogen, supplemented with 15% fetal bovine serum, 1% penicilline/streptomicine, 1% glutamine). Primary fibroblast cells migrating out of the biopsies were monitored daily and every two days the culture medium was refreshed. Confluent culture dishes were passaged, fibroblast cells were further expanded and frozen in complete F12 medium supplemented with 10% DMSO. The rest of the primary cells were expanded and used for other experiments. 2.3. Immunocytochemistry of cultured cells For immunofluorescence, cells were grown to 75% confluence and then fixed in 3% paraformaldehyde at room temperature for 25 min, permeabilized with 0.1% Triton
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X-100 in phosphate-buffered saline (PBS) for 5 min, blocked in 1% BSA in PBS for 10 min and incubated with antibodies. To detect HSPB8, we used either polyclonal (1/ 250, Abcam) or monoclonal 2H5 (1/100, Abcam) antibodies to HSPB8. Double immunostainings were performed with polyclonal HSPB8 antibody co-stained with either HSP70 (1/1000, Abcam), or ubiquitin (1/200, Dako). Alexa488- and Alexa594-conjugated secondary antibodies (Invitrogen) were used and immunofluorescence images were obtained on a confocal microscope (LSM510 Meta; Zeiss) with a 40 or 63 Plan Apo objective using LSM 510 software. For heat shock treatment (24 h after seeding), cells were incubated in a water bath at 43 °C for 30 min and were allowed to recover for 24 h at 37 °C to overcome reduced expression due to heat-induced degradation of protein before proceeding with the immunostaining. 2.4. Measurement of the mitochondrial membrane potential and mitochondrial morphology The mitochondrial membrane potential (DWm) was measured with the fluorescent lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM, Invitrogen). Patient and control fibroblast cells were stained with TMRM (200 nM) for 30 min using a 37 °C warm water bath incubator. The TMRM fluorescence was analysed on a Partec CyFlow ML cytometer (Partec, Mu¨nster, Germany) and data were analysed with the Windows Multiple Document Interface software. Cell viability was checked using propidium iodide (PI; Sigma–Aldrich); and only PInegative cells were selected for DWm analysis. Different concentrations of the ionophore valinomycin (1 lM, 200 lM, 3 mM, 12 h incubation) was tested and used to induce the collapse of the membrane potentials which served as a control for loss of DWm. In each analysis, 10,000 events were recorded and each experiment was repeated three times. For mitochondrial morphologic analysis, fibroblast cells were cultivated in 35 mm glass-bottom dishes and when cells have reached the desired confluency, the medium was removed from the dishes and 100 nM MitoTrackerR Red CMXRos (Invitrogen) prewarmed (37 °C) probe was added, and cells were incubated for 30 min. Cells were imaged on a microlens-enhanced dual spinning disk confocal live cell imaging system (UltraVIEW; PerkinElmer). During imaging, cells were maintained at 37 °C and 5% CO2. Mitochondrial morphology was quantified by using a custom-made image analysis macro in the ImageJ software (http://rsbweb.nih.gov/ij/). Briefly, segmentation of the mitochondria (creation of a binary image mask) was performed by applying the default automatic threshold of ImageJ, after application of a median filter to reduce noise (1 pixel radius), reducing background with the rolling ball algorithm (20 pixel radius) and applying a 33 top-hat filter. The shape parameters (circularity and solidity) of individual mitochondria were collected by using the imageJ Particle Analyzer on all
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objects larger than 20 pixels. Both these output parameters vary between 0 and 1, with a value of 1 describing a perfect circle. This procedure is similar to the segmentation procedure and mitochondrial morphology analysis described by [32]. The mean circularity and solidity per image field (containing 1–5 cells) was calculated and at least six image fields were used to calculate the average circularity and solidity for each fibroblast line. 2.5. Detection of caspase-3 and -7 activity in cell-based assays The Caspase-GloÒ 3/7 Assay (Promega) is a luminescent assay that measures caspase-3 and -7 activity in cell cultures. An equal volume of reagent was added to approximately 20,000 cells of patient and control fibroblasts. Luminescence was measured after 30–90 min during incubation at room temperature using a microplate luminometer (model TD-20E, Turner design) with a 96 multiwell-plate format. Luminescence data were generated by combining the in triplicate results of two caspase-3 activity experiments. Culture medium without cells was used as a blank. As a positive control for the cell death assay, we used control fibroblasts treated for 3 h with 6 lM staurosporin. 2.6. Detection of Apo-BrdU-Red incorporation in DNA of cultured cells We used the Apo-BrdU-Red in situ DNA Fragmentation Assay Kit (BioVision, Gentaur) to measure internucleosomal DNA fragmentation as a hallmark of apoptosis in mammalian cells. Fibroblast cells were fixed with 3% formaldehyde in PBS and the DNA fragmentation assay was performed according to the manufacturer’s protocol. As a positive control for the cell death assay, we used control fibroblasts treated for 3 h with 3 lM staurosporin. 2.7. Western blotting Cells were lysed in NP40 lysis buffer (250 mM NaCl, 1 mM EDTA, 20 mM HEPES pH 7.9, 1% NP40, 20 mM b-glycerophosphate, 10 mM sodium fluoride, 4 mM sodium orthovanadate, 400 lg/ml sodium pyrophosphate, and 2 mM DDT) supplemented with protease inhibitor cocktail (Roche) for 20 min on ice and cleared by centrifugation. Cell extracts were boiled for 5 min and equal concentrations of cell lysates were resolved on a SDS– NuPage gel. The gel was transferred to a nitrocellulose membrane and processed for Western blotting as described previously [27,33]. For analysis of apoptosis, we used: cleaved-caspase 3, total-caspase 3, cleaved-caspase 9, total-caspase 9, cleaved-PARP and total-PARP (all antibodies were obtained from Cell Signalling and used at 1/1000 dilution). For analysis of protein expression, we used commercial available monoclonal HSPB8 antibody (1/100, Abcam) and anti-b-Actin (Sigma, 1/5000).
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2.8. Light and transmission electron microscopy Araldite blocks of glabrous dermal punch of two patients and controls were used for electron microscopy. Immersion fixation was achieved with 4% neutral buffered glutaraldehyde followed by 2% buffered osmium tetroxide and embedding in araldite [34]. Semithin sections were obtained with a Reichert Jung ultramicrotome (Leica, Wein, Austria) and collected on glass slides for light microscopy. Dermal myelinated nerve bundles were identified under the light microscope and were then trimmed and cut into ultrathin sections collected on copper grids. Sections were contrasted with uranyl acetate and lead citrate, and 34 cutaneous nerve bundles (6 nerves from patients; 28 nerves from control) were analyzed by a FEI CM10 transmission electron microscope (Philips, Eindhoven, The Netherlands) equipped with a goniometric coordinator. 3. Results 3.1. Distal HMN fibroblasts show transient HSPB8-positive protein aggregates In 2004, we reported that the HSPB8-K141N mutation results in perinuclear and cytoplasmic aggregates in transfected COS1 cells, and that these aggregates sequestered the HSPB1 protein [1]. As these experiments were based on transient transfection of mutant HSPB8 constructs, we could not exclude that the aggregates were due to possible overexpression of the mutant transcript. To exclude potential overexpression artifacts and to study the effect of mutant HSPB8 in dHMN patient cells we obtained skin biopsies from two related dHMN patients with the HSPB8-K141N mutation. Our findings were compared with skin biopsies from three unrelated healthy controls. From the skin biopsies we generated cultured fibroblasts, according to methods described. Gross morphological differences were not observed between cultured fibroblasts from the patients and control individuals. However, when we studied the cells by immunofluorescence microscopy using a HSPB8 directed antibody staining, every examined early passage primary fibroblast cell from dHMN patients showed multi-foci protein precipitates distributed throughout the cytoplasm. These HSPB8-positive aggregates were mainly detected in the early passage cultures (1–4 weeks) (Fig. 1A, patient). Primary fibroblasts from control individuals cultured under the same conditions mostly showed a homogeneous distribution of HSPB8-staining throughout the cytoplasm and nucleus (Fig. 1A, control). In later passage cultures (5–12 weeks), the mutant HSPB8 protein precipitates in patients’ cells coalesced into fewer cytoplasmic and/or perinuclear HSPB8positive aggregates and in some cells the protein accumulations were seen clustered around unknown structures in the cell (Fig. 1B). While protein aggregates in the early passage fibroblasts of dHMN patient were smaller in size and observed in every examined cell, those in the late passages were larger in diameter and scarcely observed (Fig. 1C).
After two months in culture, the percentage of aggregatepositive cells was still significantly higher in patients’ cells (data of three parallel experiments, n = 3 200 cells were counted: control HSPB8 wild type (WT) = 9.8% ± 1.52; patient 1-K141N = 37.6% ± 3, p-value t-test = 2.8 10 4; patient 2-K141N = 32.5% ± 14.7, p-value = 8.9 10 3). Next, we further examined this striking reduction in aggregate positive cells with increasing time in culture (Fig. 1C). To check whether the HSPB8 protein aggregates were targeted for degradation, we performed co-immunostainings with HSP70 and ubiquitin antibodies in dHMN fibroblasts and found that HSPB8-positive aggregates show enhanced HSP70 and ubiquitin protein localization (Fig. 2). These results indicate that fibroblasts containing HSPB8-protein aggregates are capable of recruiting the cellular machinery for elimination of either refolded or degraded misfolded HSPB8 proteins. However, we cannot exclude that selection of aggregate-negative cells in culture had contributed to the decrease in the percentage of aggregate-positive cells over time. Subsequently, we tested the effect of applying a heat shock (HS) stress on these cultured cells. We found that in the patients’ early passage cells multi-foci mutant HSPB8 precipitates coalesced into larger aggregates upon HS-stimulation, while control cells showed a homogenous upregulation of HSPB8 protein expression (Fig. 1D +HS). Protein expression of HSPB8 shown by Western blot was increased after heat shock, as was previously described [35,20], but no drastic changes were observed between patient and control fibroblasts (Fig. 1E). This study shows that upon heat shock stress the HSPB8 precipitates coalesced into larger protein aggregates which were subsequently cleared out as the passage time in cultures increased. Presumably mutant HSPB8 protein aggregates are cleared out with time in culture suggesting no cumulative effects and no major consequence in dividing fibroblast cells. 3.2. Mitochondrial membrane potential is reduced in early passage mutant fibroblasts Modulation of mitochondrial function and morphology is critical in neurodegenerative disorders [36]. Accumulation of misfolded proteins has been shown to disrupt mitochondrial function [37,38], plausibly by inducing defects in mitochondrial membrane potential (DWm) [39,40]. We first investigated whether there were changes in mitochondrial morphology by using live-cell imaging with cell-permeant MitoTracker Red CMXRos dye. The shape of the mitochondria was analyzed by measuring their average circularity and solidity. No differences could be detected in the mitochondrial shape descriptors between the control and patient fibroblasts (Supplementary figure 1). Absence of significant alterations in mitochondrial morphology does not exclude potential impairment of mitochondrial function. We further tested whether the fibroblast cells from patients exhibited a defect in mitochondrial energy metabolism. We measured DWm using a
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Fig. 1. HSPB8-K141N protein aggregates in dHMN skin fibroblasts. Primary fibroblast cells from skin biopsy of two patients and control persons were processed for immunofluorescence analysis. (A–B) The patients’ cells exhibit misfolded HSPB8 positive aggregates (patient, A and B, early and late passage). Such protein accumulations were absent in cells from control individuals (control, A and B). (C) The number of aggregate positive cells with increasing passage time in culture. (D) Heat shock stimulation of patients’ early passage cells (patient, D, +HS). Heat shock stimulation of control cells (control, D, +HS). (E) Western blot analysis of HSPB8 protein expression in patients and controls cell extracts. Selected images are representative for all patients and controls samples analyzed. No heat shock ( HS), with heat shock (+HS), nucleus is shown in blue, scale bar = 20 lm. Normalization was performed with b-actin.
fluorescent lipophilic cationic dye (TMRM) that accumulates within healthy polarized mitochondria. TMRM red fluorescence was measured by flow cytometry and analysis was performed in viable propidium iodide negative cells only. The depolarizing drug valinomycin was used as a positive control for loss of DWm (Fig. 3A, valinomycin). In early passage control cells, a high DWm (geometric mean value of 2 control samples = 397.1) was found demonstrating that the majority of the mitochondria were well polarized over the inner mitochondrial membrane (Fig. 3B and C, control 4 and 5). In contrast, when early passage patients’ cells were stained with TMRM, DWm was significantly lower (geometric mean value 2 patient samples = 272.6), suggesting that a significant portion of patient’s mitochondria were not adequately polarized (Fig. 3B and C, patient 1 and 2). (Mean data of three experiments, n = 3 10,000 cells per genotype were analysed: HSPB8-WT fibroblast control 4 = 433.49 ± 34; control 5 = 360.7 ± 18; patient 1-K141N = 251.6 ± 13,
p-value t-test = 5.8 10 3; patient 2-K141N = 293.5 ± 7, p-value = 1.5 10 2). When the same experiment was performed in late passage cells (after three months in culture) significant differences in DWm between patient and control groups were not observed (data not shown). These results suggest that the anomalous effects of mutant HSPB8 are well tolerated in dividing fibroblast cells. 3.3. Distal HMN fibroblasts do not show a major increase in apoptosis compared to control cells In many neurodegenerative diseases accumulation of misfolded proteins has been linked to cellular dysfunction and cell death [38,41]. Here, we investigated the relative survival of patient fibroblasts carrying the HSPB8K141N mutation by measuring the caspase activities. The apoptosis assays were only performed in late passage cells after two months in culture and not in early passage cells, since the early passage cultures were no longer available.
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Fig. 2. Co-localization of mutant HSPB8 protein aggregates with HSP70 and ubiquitin. Patient and control fibroblast cells were immunostained with antibodies against either HSP70 (green) or Ubiquitin (green) and HSPB8 (red). (A) Control fibroblast cells and (B) patient fibroblast cells both immunostained with HSP70 and HSPB8 antibodies. (C) Control fibroblast cells and (D) patient fibroblast cells both immunostained with antibodies to ubiquitin and HSPB8. Selected images are representative for all patients and controls samples analysed. Nucleus is shown in blue, Scale bar = 10 lm.
We observed an increase of caspase 3/7 activity in fibroblasts of patient 2 but not of patient 1 as compared to control fibroblasts (Fig. 4A, patient 2 vs. control 3 pvalue = 0.037, patient 2 vs. control 5 p-value = 0.057. Patient 1 vs. control 3 p-value = 0.138, patient 1 vs. control 5 p-value = 0.247). In addition, we investigated the distribution of internucleosomal DNA fragmentation as a hallmark of apoptosis [42]. Staurosporin treatment was used
as a positive control to induce apoptosis (Fig. 4B). Control fibroblasts were Br-dUTP negative (Fig. 4C). The patients’ fibroblast cultures contained more Br-dUTP positive cells compared to control cultures (Fig. 4D and E). Furthermore, protein levels of three well established apoptotic markers were evaluated in late passage cells after three months in culture: cleaved-caspase 3, cleaved-caspase 9 and cleaved-poly (ADP-ribose) polymerase (PARP)
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Fig. 3. Loss of mitochondrial membrane potential in patient’s fibroblasts. Mitochondrial membrane potential (DWm) was measured in cultured patients and controls fibroblasts. (A) Valinomycin treated cells were used as a positive control for loss of DWm. (B and C) TMRM histogram analysis of DWm of 2 patients and 2 control fibroblast cells. (D) Statistical analysis (DWm geometric mean and stdev) was performed on three different experiments.
[43,44]. There was no clear evidence of accumulation of these apoptotic markers in either patients or controls cell lysates in two different experiments (Fig. 5). Taken together, these results indicate that the HSPB8-K141N mutation could elevate the vulnerability to pro-apoptotic stimuli in primary fibroblast cultures, although not all of used apoptosis markers confirm these results. 3.4. Cutaneous nerves show axonal loss in dHMN patients Since we were unable to obtain post-mortem spinal cord or motor nerve biopsy samples from dHMN patients with the HSPB8-K141N mutation to test the presence of intracellular protein aggregates, we studied skin biopsy cutaneous nerves from two dHMN patients [28] by electron microscopy. This approach constitutes a minimal invasive procedure and is also used to monitor the progression of the neuropathy and regeneration of nerve fibers [45,46]. Electron microscopy of cutaneous nerve fascicles revealed a decreased number of myelinated and unmyelinated axons in both patients (Fig. 6A and C), in comparison with agematched control individuals such as illustrated in a dermal nerve bundle of a 53-year old control (Fig. 6B). Few formations with slight axonal swellings, suggestive of axonal regeneration sprouting (not shown), and a thinly myelinated (regenerated) axon were detected (Fig 6C). Denervated Schwann cells forming collagen pockets (Fig 6D) and large bundles of endoneurial collagen fibrils (Fig 6A), were observed. Ultrastructural analysis did not reveal obvious protein inclusions, neither did it reveal alterations of the mitochondrial morphology, nor onion-bulb-like
formations in the patients’ cutaneous nerve fascicles. These results suggest that the mutation in HSPB8 affects sensory axons but only to a minor extent. 4. Discussion HSPB8 belongs to the family of small heat shock proteins. These proteins are multifunctional molecular chaperones that participate in multiple cellular processes [47–49]. Although HSPB8 is expressed in many tissues [20,50], the expression level is higher in the peripheral nervous system [1,35] which explains why mutations in HSPB8 cause dHMN and CMT2 neuropathy [1,10]. Previously, we demonstrated that rodent motor neuron cultures transiently expressing mutant HSPB8 protein resulted in distal neurite degeneration without apoptosis [27]. In a former work, we showed that overexpression of mutant HSPB8 resulted in intracellular protein aggregates [1]. Here, we show that early passage fibroblast cells from two related dHMN patients have an accumulation of HSPB8 protein aggregates and a decrease in mitochondrial membrane potential. These protein aggregates were cleared out after three months in culture and patient cells became indistinguishable from controls. We also observed that under stress conditions endogenous mutant HSPB8 protein induced intracellular protein aggregates in cultured skin fibroblasts from the dHMN patients. Such accumulation of protein was not seen in three fibroblasts from unrelated healthy individuals. Heat shock stress stimulation of patients’ early passage cells resulted in larger accumulations of HSPB8 positive
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Fig. 4. Patient fibroblast cells do not elicit marked apoptosis. Cell death assay of: (A) dHMN patients and controls fibroblast cells. As positive control, cells were treated with staurosporin, and cell death luminescence was measured. Data are presented as means ± stdev of three different experiments. (B–D) Apoptosis assay of patients and control fibroblast cells were processed for DNA fragmentation followed by immunofluorescence. (B) Control fibroblasts were treated with staurosporin as positive control for cell death and immunostained with HSPB8 antibody (green) and processed for DNA fragmentation (red). (C) Control fibroblasts. (D) Patient fibroblasts. (E) The quantification of DNA fragmentation in patient and control fibroblasts. Selected images are representative for all patient and control samples analyzed. Scale bar = 30 lm. Statistical significance denoted with a star (*p-value <0.05) was determined using Student’s t-test.
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Fig. 5. Mutant fibroblasts do not result in increased activation of apoptosis. Activation of enzymes implicated in initiation and execution of apoptosis was examined by Western blot of cleaved (17 and 19 kDa) and total caspase 3 (35 kDa), cleaved (35 and 37 kDa) and total caspase 9 (47 kDa) and cleaved (89 kDa) and total PARP (116 kDa) in patients and controls fibroblast cells. Selected images are representative for two different western blot experiments. Normalization was performed with b-actin.
precipitates into aggresome-like structures and not in a homogenous upregulation of HSPB8 protein as observed in control cells. An accumulation of more protein aggregates suggests that the mutant protein is associated with a defect in stress-induced molecular chaperones that are considered as vital for cell viability. Evidently, in the endoplasmic reticulum proteins that fail to fold are destroyed.
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Such misfolded proteins are transported to the cytosol where ubiquitination and finally proteasomal proteolysis dispense with the unwanted polypeptides [51]. Our data showed that the HSPB8 protein aggregates tested positive for both HSP70 and ubiquitin, suggesting that the protein accumulations are being degraded by proteasomal proteolysis. We cannot exclude the possibility of other mechanisms for intracellular protein degradation being activated. In addition to its canonical function as a molecular chaperone, HSPB8 acts in association with the cochaperone BAG3 to target protein aggregates for destruction via autophagy [52–54]. However, in a recent report it was shown that overexpression of the HSPB8-K141N mutant protein failed to stimulate autophagy [55]. In any case, the observation that the number of mutant HSPB8 cells with protein aggregates decreases with an increase in passage time, points to a stress induction at the start of the culture that causes mutant HSPB8 to aggregate, and a subsequent clearance of these aggregates during the time in culture or alternatively, a selection of aggregate-free cells. We can speculate that stress caused by the biopsy procedure and adaptation to cell culture conditions caused a transient HSPB8 upregulation, which resulted in aggregation of the mutant protein. Refolding of misfolded protein could deplete cellular energy pools that are needed for normal cellular functions, especially in a confined axoplasmic region. Mitochondrial dysfunction plays a pivotal role in contributing to defective energy metabolism and neuronal degeneration [56]. We tested whether the fibroblast cells from patients exhibit any defect in mitochondrial energy metabolism using TMRM, a mitochondria transmembrane potential (DWm) marker. We did not observe obvious changes in mitochondrial morphology but detected a significant reduction in DWm in the patients’ fibroblast cells as compared to control cells. These data suggest that dHMN fibroblasts have more depolarized mitochondria than control fibroblasts. An extrapolation of the observations made in patients’ cultured fibroblasts to the nervous system should be interpreted with caution but might explain some of the findings in the nervous system, especially in the motor neurons with long axons. It is possible that a reduction of the mitochondrial DWm is well tolerated in the cells without axons, but could become detrimental in the cells with long axons. Accumulation of depolarized mitochondria in a late onset disease could disrupt cellular homeostasis and trigger synaptic and axoplasmic dysfunctions [38]. In the present study, cells of patient primary fibroblast show increased incidence of apoptosis based on some of the markers we used. In a former work however, we studied the significance of HSPB8 mediated cell death in primary rodent ventral horn cultures after four days of mutant protein transduction and found cell type specific neurite defects, but no signs of defective apoptosis in all studied cell types [27]. Given that axonal and not neuronal loss is the first manifestation of the disease in patients [28,57], we do not assume that upregulation of apoptotic signaling
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Fig. 6. Evidence of axonal loss in skin biopsy of patients with the HSPB8-K141N mutation. Electron micrographs of cutaneous nerve fascicles. General views revealing a decreased number of myelinated and unmyelinated axons in both patients (A and C) in comparison with a control individual (B). Note obvious endoneurial collagen formation (arrows) in (A) and a thinly myelinated (regenerating) axon (arrow) (C). Higher magnification of denervated Schwann cells with collagen pockets (arrows) of the nerve illustrated in C (D) while several normal unmyelinated axons () within Schwann cell (arrows) cytoplasms, are shown in (E). Scale bars = 1 lm.
is a primary mechanism for mutant HSPB8 pathogenicity. We consider that the increased susceptibility to apoptosis in the mutant HSPB8 cultures is a secondary and more general effect in the mutant HSPB8 fibroblasts that results from their defective capacity to handle cellular stress. Overall, our results demonstrate that although patients’ fibroblast cells exhibited subtle signs of stress, these cells were able to tolerate the anomalous effects of mutant HSPB8. We speculate that accumulation of such subtle signs of stress might be well tolerated in the neuronal cell body but plausibly could contribute to the process of axonal loss in dHMN. Corroborating our reasoning, it was suggested in a recent report that if a neuropathy takes years to develop, the mutants that cause such a neuropathy would be expected to have relatively subtle defects and only with time are the cumulative effects of the mutation on cellular function likely to become fully apparent [58]. Notably, the lack of dramatic effects on apoptosis in patients’
cells is in accordance with the dHMN disease phenotype, being debilitating but not life threatening. As dHMN is predominantly a motor neuropathy, it would be ideal to study motor nerves but we were unable to obtain motor nerve autopsy material. Motor nerves are also difficult to obtain by biopsy because it would cause a motor deficit in the corresponding muscles. The only nerves that were available for investigation were sensory cutaneous nerves in a skin biopsy specimen from the two patients carrying the HSPB8 K141N mutation. Ultrastructural analysis carried out by transmission electron microscopy [28] revealed that patients’ cutaneous nerves showed clear signs of axonal loss and slight axonal regeneration. We cannot exclude the possibility that these findings might be due to age-related changes [59] although such ultrastructural findings were not detected in the investigated control skin biopsies. We did not observe any obvious protein inclusion or abnormal mitochondrial shape in the skin
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biopsies from the patients. Overall, our results indicate that mutant HSPB8 induces only minor degeneration of sensory nerve axons in dHMN patients’ cutaneous nerves. This confirms that, although the brunt of the injury in motor neuropathies falls on the motor neurons, some minor sensory involvement can be detected. Of note is that the K141N mutation is not only associated to dHMN but also to CMT2 phenotype [10]. By using skin biopsies and derived fibroblasts to study dHMN, a disease that primarily affects motor neurons, we are obviously using a suboptimal disease model, especially since we have shown that the pathogenic effect of the HSPB8 mutation is specifically affecting motor neurons [27]. In this regard, ultrastructural examination of cutaneous nerve fascicles allows examining sensory nerves rather than motor nerves (Fig. 6). In addition, since biopsies of the relevant patients are relatively scarce, it was not feasible to include larger numbers of individuals in this study to further ascertain our findings. Moreover, during the course of this study we were confronted with the observation that in the initial passages of the fibroblast cultures, the majority of cells showed mutant HSPB8 aggregates, whereas after some time in culture the prevalence of HSPB8 aggregate positive fibroblasts decreased unexpectedly. Because it was not possible to resample the same patients, mainly for deontological reasons, we could not reassess the patient fibroblasts in their initial state to replicate our experiments and to further explore this phenomenon experimentally. However, there are also benefits in using this approach compared to alternative strategies. (i) It allows the examination of patient’s endogenous mutant proteins without the complications of overexpression artifacts (Fig. 1). (ii) It can reveal damage or morphological alterations in small dermal nerve fascicles (Fig. 6), and in contrast to conventional peripheral nerve biopsy a skin biopsy is less invasive and is easier to perform [46]. (iii) Skin biopsy fibroblasts can be reprogrammed to generate patient-specific induced pluripotent stem cells and neural progenitor cells (iPS and NPC), which can be used to reveal novel biological insights which can, in turn, lead to clinical benefits [60]. In conclusion our work demonstrates that early passage primary fibroblasts from dHMN patients show abnormal changes including the presence of protein aggregates, a decrease of the inner mitochondrial potential and a reduction in cell viability. These defects were not observed in late passage primary cells, which emphasizes the complexity and inadequacy of studying a neuropathy by using nonneuronal patient material. Direct reprogramming of the patients’ fibroblasts to iPS cell-derived neurons could provide a platform to understand the effect of mutant sHSP’s in human motor neurons and could reveal reliable biomarkers for disease prevention strategies. Acknowledgments We are grateful to the patients and control individuals who provided nerve and skin biopsies. The authors thank
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G. Munteanu, K.G. Claeys, C. Michiels, N. Cools, M. Lenjuo, V. De Corte, B. Van Loo, J. Smet and N. Festjens for technical assistance. We thank J. Wauters, J. Lambert, R. Bendorf, K. Verhoeven, V. Van Tendeloo for discussions and exchange of expertise. We thank L. Svensson, J. Van Daele, D. De Rijck, L. De Wit and I. Bats for technical assistance with electron and confocal microscopy. This work was supported by the Methusalem and Hercules (AUAH-09-001) programs of the Flemish Government, the Fund for Scientific Research (FWO-Flanders), the Medical Foundation Queen Elisabeth, the “Association Belge contre les Maladies Neuromusculaires”, the American Muscular Dystrophy Association, and the Interuniversity Attraction Poles P6/43 program of the Belgian Federal Science Policy Office. A.H. is supported by a Ph.D. fellowship of the Institute for Science and Technology (IWT). J.I. is postdoctoral researcher supported by the FWOFlanders. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.nmd.2012.04.005. References [1] Irobi J, Van Impe K, Seeman P, et al. Hot-spot residue in small heatshock protein 22 causes distal motor neuropathy. Nat Genet 2004;36:597–601. [2] Evgrafov OV, Mersiyanova I, Irobi J, et al. Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. Nat Genet 2004;36:602–6. [3] Rossor AM, Kalmar B, Greensmith L, Reilly MM. The distal hereditary motor neuropathies. J Neurol Neurosurg Psychiatry 2012;83:6–14. [4] Kijima K, Numakura C, Goto T, et al. Small heat shock protein 27 mutation in a Japanese patient with distal hereditary motor neuropathy. J Hum Genet 2005;50:473–6. [5] Houlden H, Laura M, Wavrant-DeVrieze F, et al. Mutations in the HSP27 (HSPB1) gene cause dominant, recessive, and sporadic distal HMN/CMT type 2. Neurology 2008;71:1660–8. [6] James PA, Rankin J, Talbot K. Asymmetrical late onset motor neuropathy associated with a novel mutation in the small heat shock protein HSPB1 (HSP27). J Neurol Neurosurg Psychiatry 2008;79:461–3. [7] Luigetti M, Fabrizi GM, Madia F, et al. A novel HSPB1 mutation in an Italian patient with CMT2/dHMN phenotype. J Neurol Sci 2010;298:114–7. [8] Capponi S, Geroldi A, Fossa P, et al. HSPB1 and HSPB8 in inherited neuropathies: study of an Italian cohort of dHMN and CMT2 patients. J Peripher Nerv Syst 2011;16:287–94. [9] Mandich P, Grandis M, Varese A, et al. Severe neuropathy after diphtheria-tetanus-pertussis vaccination in a child carrying a novel frame-shift mutation in the small heat-shock protein 27 gene. J Child Neurol 2010;25:107–9. [10] Tang BS, Zhao GH, Luo W, et al. Small heat-shock protein 22 mutated in autosomal dominant Charcot-Marie-Tooth disease type 2L. Hum Genet 2005;116:222–4. [11] Kolb SJ, Snyder PJ, Poi EJ, et al. Mutant small heat shock protein B3 causes motor neuropathy: utility of a candidate gene approach. Neurology 2010;74:502–6.
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J. Irobi et al. / Neuromuscular Disorders 22 (2012) 699–711
[12] Harding AE, Thomas PK. Hereditary distal spinal muscular atrophy. A report on 34 cases and a review of the literature. J Neurol Sci 1980;45:337–48. [13] Saporta AS, Sottile SL, Miller LJ, et al. Charcot-Marie-Tooth disease subtypes and genetic testing strategies. Ann Neurol 2011; 69:22–33. [14] Dierick I, Baets J, Irobi J, et al. Relative contribution of mutations in genes for autosomal dominant distal hereditary motor neuropathies: a genotype-phenotype correlation study. Brain 2008;131:1217–27. [15] Haslbeck M, Franzmann T, Weinfurtner D, Buchner J. Some like it hot: the structure and function of small heat-shock proteins. Nat Struct Mol Biol 2005;12:842–6. [16] Drew AP, Blair IP, Nicholson GA. Molecular genetics and mechanisms of disease in distal hereditary motor neuropathies: insights directing future genetic studies. Curr Mol Med 2011. [17] Dierick I, Irobi J, De Jonghe P, Timmerman V. Small heat shock proteins in inherited peripheral neuropathies. Ann Med 2005;37:413–22. [18] Rothman JE, Schekman R. Molecular mechanism of protein folding in the cell. Cell 2011. [19] Carra S, Sivilotti M, Chavez Zobel AT, Lambert H, Landry J. HspB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells. Hum Mol Genet 2005;14:1659–69. [20] Chowdary TK, Raman B, Ramakrishna T, Rao CM. Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperonelike activity. Biochem J 2004;381:379–87. [21] Wilhelmus MM, Boelens WC, Otte-Holler I, et al. Small heat shock proteins inhibit amyloid-beta protein aggregation and cerebrovascular amyloid-beta protein toxicity. Brain Res 2006;1089:67–78. [22] Wilhelmus MM, Otte-Holler I, Wesseling P, et al. Specific association of small heat shock proteins with the pathological hallmarks of Alzheimer’s disease brains. Neuropathol Appl Neurobiol 2006;32:119–30. [23] Bera S, Thampi P, Cho WJ, Abraham EC. A positive charge preservation at position 116 of alpha A-crystallin is critical for its structural and functional integrity. Biochemistry 2002;41:12421–6. [24] Kasakov AS, Bukach OV, Seit-Nebi AS, Marston SB, Gusev NB. Effect of mutations in the beta5–beta7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11). FEBS J 2007;274:5628–42. [25] Litt M, Kramer P, LaMorticella DM, et al. Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystallin gene CRYAA. Hum Mol Genet 1998;7:471–4. [26] Vicart P, Caron A, Guicheney P, et al. A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat Genet 1998;20:92–5. [27] Irobi J, Almeida-Souza L, Asselbergh B, et al. Mutant HSPB8 causes motor neuron-specific neurite degeneration. Hum Mol Genet 2010;19:3254–65. [28] Timmerman V, Raeymaekers P, Nelis E, et al. Linkage analysis of distal hereditary motor neuropathy type II (distal HMN II) in a single pedigree. J Neurol Sci 1992;109:41–8. [29] Pedrini S, Sau D, Guareschi S, et al. ALS-linked mutant SOD1 damages mitochondria by promoting conformational changes in Bcl2. Hum Mol Genet 2010;19:2974–86. [30] Caspersen C, Wang N, Yao J, et al. Mitochondrial Abeta: a potential focal point for neuronal metabolic dysfunction in Alzheimer’s disease. FASEB J 2005;19:2040–1. [31] Hauptmann S, Keil U, Scherping I, et al. Mitochondrial dysfunction in sporadic and genetic Alzheimer’s disease. Exp Gerontol 2006;41:668–73. [32] Koopman WJ, Verkaart S, Visch HJ, et al. Inhibition of complex I of the electron transport chain causes O2 -mediated mitochondrial outgrowth. Am J Physiol Cell Physiol 2005;288:C1440–50. [33] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76:4350–4.
[34] Ceuterick C, Martin JJ. Electron microscopic features of skin in neurometabolic disorders. J Neurol Sci 1992;112:15–29. [35] Kirbach BB, Golenhofen N. Differential expression and induction of small heat shock proteins in rat brain and cultured hippocampal neurons. J Neurosci Res 2011;89:162–75. [36] Kieper N, Holmstrom KM, Ciceri D, et al. Modulation of mitochondrial function and morphology by interaction of Omi/ HtrA2 with the mitochondrial fusion factor OPA1. Exp Cell Res 2010;316:1213–24. [37] Winklhofer KF, Tatzelt J, Haass C. The two faces of protein misfolding: gain- and loss-of-function in neurodegenerative diseases. EMBO J 2008;27:336–49. [38] Schon EA, Manfredi G. Neuronal degeneration and mitochondrial dysfunction. J Clin Invest 2003;111:303–12. [39] Correia SC, Santos RX, Perry G, et al. Mitochondria: the missing link between preconditioning and neuroprotection. J Alzheimers Dis 2010;20(Suppl. 2):S475–85. [40] Balaban RS, Nemoto S, Finkel T. Mitochondria, oxidants, and aging. Cell 2005;120:483–95. [41] Hoffstrom BG, Kaplan A, Letso R, et al. Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins. Nat Chem Biol 2010;6:900–6. [42] Saraste A, Pulkki K. Morphologic and biochemical hallmarks of apoptosis. Cardiovasc Res 2000;45:528–37. [43] Kroemer G, Galluzzi L, Vandenabeele P, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 2009;16:3–11. [44] Vanden Berghe T, Denecker G, Brouckaert G, et al. More than one way to die: methods to determine TNF-induced apoptosis and necrosis. Methods Mol Med 2004;98:101–26. [45] Ceuterick-De GC, De Jonghe P, Timmerman V, et al. Infantile demyelinating neuropathy associated with a de novo point mutation on Ser72 in PMP22 and basal lamina onion bulbs in skin biopsy. Pathol Res Pract 2001;197:193–8. [46] Li J, Bai Y, Ghandour K, et al. Skin biopsies in myelin-related neuropathies: bringing molecular pathology to the bedside. Brain 2005;128:1168–77. [47] Arrigo AP, Paul C, Ducasse C, et al. Small stress proteins: novel negative modulators of apoptosis induced independently of reactive oxygen species. Prog Mol Subcell Biol 2002;28:185–204. [48] Charette SJ, Landry J. The interaction of HSP27 with Daxx identifies a potential regulatory role of HSP27 in Fas-induced apoptosis. Ann N Y Acad Sci 2000;926:126–31. [49] Huot J, Houle F, Spitz DR, Landry J. HSP27 phosphorylationmediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res 1996;56:273–9. [50] Benndorf R, Sun X, Gilmont RR, et al. HSP22, a new member of the small heat shock protein superfamily, interacts with mimic of phosphorylated HSP27 ((3D)HSP27). J Biol Chem 2001;276:26753–61. [51] Lilley BN, Ploegh HL. A membrane protein required for dislocation of misfolded proteins from the ER. Nature 2004;429:834–40. [52] Crippa V, Sau D, Rusmini P, et al. The small heat shock protein B8 (HspB8) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS). Hum Mol Genet 2010;19:3440–56. [53] Arndt V, Dick N, Tawo R, et al. Chaperone-assisted selective autophagy is essential for muscle maintenance. Curr Biol 2010;20:143–8. [54] Carra S, Seguin SJ, Landry J. HspB8 and Bag3: a new chaperone complex targeting misfolded proteins to macroautophagy. Autophagy 2008;4:237–9. [55] Kwok AS, Phadwal K, Turner BJ, et al. HspB8 mutation causing hereditary distal motor neuropathy impairs lysosomal delivery of autophagosomes. J Neurochem 2011. [56] Eckert A, Keil U, Marques CA, et al. Mitochondrial dysfunction, apoptotic cell death, and Alzheimer’s disease. Biochem Pharmacol 2003;66:1627–34.
J. Irobi et al. / Neuromuscular Disorders 22 (2012) 699–711 [57] Timmerman V, Beuten J, Irobi J, et al. Distal hereditary motor neuropathy type II (distal HMN type II): phenotype and molecular genetics. Ann N Y Acad Sci 1999;883:60–4. [58] Holzbaur EL, Scherer SS. Microtubules, axonal transport, and neuropathy. N Engl J Med 2011;365:2330–2.
711
[59] Timmerman V, De Jonghe P, Simokovic S, et al. Distal hereditary motor neuropathy type II (distal HMN II): mapping of a locus to chromosome 12q24. Hum Mol Genet 1996;5:1065–9. [60] Han SS, Williams LA, Eggan KC. Constructing and deconstructing stem cell models of neurological disease. Neuron 2011;70:626–44.